Dried formulations of nanoparticle-coated capsules

ABSTRACT

A method of producing a dried formulation for an active substance such as a drug compound is described. The method involves dispersing a discontinuous phase (e.g. an oil-based or lipidic medium) comprising the active substance into a continuous phase (e.g. water) so as to form a two-phase liquid system comprising droplets of said discontinuous phase, allowing nanoparticles to congregate at the phase interface at the surface of the droplets such that at least one layer of nanoparticles coat the droplets and thereby provide sufficient structural integrity to the droplets to enable the subsequent removal of the continuous phase, and thereafter removing the continuous phase from the nanoparticle-coated droplets to produce a dried formulation.

This application is a continuation of U.S. Ser. No. 13/653,909, filedOct. 17, 2012, which is a continuation of U.S. Ser. No. 12/902,769,filed Oct. 12, 2010, now U.S. Pat. No. 8,303,992, which is acontinuation of U.S. Ser. No. 11/916,570, filed Dec. 5, 2007, nowabandoned, which is a 371 filing of PCT/AU2006/000771, filed Jun. 7,2006 which claims priority from Australian Patent Application No.2005902937, filed Jun. 7, 2005. These prior applications areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the encapsulation by nanoparticles of aliquid droplet or a lipid vesicle to form a stable capsule.

BACKGROUND OF THE INVENTION

The development of new forms of active substances such as drug compoundsand pesticides, as well as a desire to increase the efficacy of existingsubstances, has created a need to develop new and effective ways ofdelivering substances to their appropriate targets. It is likely thatmany potentially useful active substances have not been commercialisedbecause of inadequate formulation. In many cases, the inability toformulate the active substance into a deliverable form could simply bedue to solubility problems.

Although useful as vehicles for the delivery of active substances, mostemulsions and liposomes are limited by the fact they arethermodynamically unstable and, generally, over time, will coalesce andmay eventually separate into two distinct liquid phases (emulsions) orwill degrade and release the fluid-filled core into the surroundingmedia (liposomes). This instability is exacerbated in veterinary andpharmacological applications since the vehicles are used undercircumstances (e.g. increased salt (electrolyte) or variations in pH)which put a severe strain on the vehicle structure. The degradation ofvehicles containing active substances is undesirable since considerabletime and effort is spent in formulating the delivery system. In theveterinary, pharmaceutical and nutriceutical industries in particular,if vehicle stability is compromised, the bioavailability of the activesubstance may be affected.

Particle stabilised emulsions are known, however, the stability of theresulting capsules remains poor over a period of time. This means thatit is difficult to transport the capsules over long distances and it isdifficult to store the capsules for a delayed time of use. As thecapsules degrade, the active substance (e.g. a drug compound or apesticide) within the capsules can leach out, or may be released withoutcontrol. Leaching or uncontrolled release of active substances can posea serious problem in the delivery of certain drugs in the body, sinceone intent of the encapsulation process is to shield healthy cells fromthe drug's toxicity and prevent the drug from concentrating invulnerable tissues (e.g. the kidneys and liver).

Existing preparations of particle stabilised vehicles (capsules) areusually dispersed in a liquid in order that the capsules can bedelivered to the body as a liquid suspension. These liquid formulationsusually have a low active substance content to liquid ratio and, inaddition, during storage or transport, there is a risk of microbialgrowth in the liquid which can cause serious infections or spoilage.

A further problem is coalescence of the capsules to form capsules withan increased diameter. Larger capsules are less stable over time, andlarger capsules cannot be delivered to some areas where the diameter ofthe capsule will not be permitted (e.g. capillaries in the body).Further to this, active substance release profiles are correlated withinterfacial surface area. It is important, therefore, that capsule sizeremain constant in order that the release profile of the activesubstance is maintained.

Accordingly, it is an object of the present invention to provide acapsule for the delivery and/or dry storage of an active substance whichhas a relatively long shelf-life and is therefore easy to store ortransport and may have a reduced risk of microbial growth duringstorage.

SUMMARY OF THE INVENTION

A method of producing a dried formulation for an active substance, saidmethod comprising the steps of:

-   -   (i) dispersing a discontinuous phase comprising an active        substance into a continuous phase so as to form a two-phase        liquid system comprising droplets of said discontinuous phase,        each of said droplets having, at its surface, a phase interface;    -   (ii) allowing nanoparticles provided to said two-phase liquid        system to congregate at the phase interface to coat said surface        of the droplets in at least one layer of said nanoparticles,        wherein said at least one layer of nanoparticles provides        sufficient structural integrity to the droplets to enable the        subsequent removal of the continuous phase; and    -   (iii) removing the continuous phase from the nanoparticle-coated        droplets to produce a dried formulation.

The discontinuous phase may be dispersed in the continuous phase to forma two-phase liquid system (e.g. an emulsion) by any of the methods wellknown to persons skilled in the art (e.g. by homogenisation).

Preferably, the discontinuous phase is an oil-based or lipidic medium(e.g. a phospholipid preparation), and the continuous phase is aqueous.

However, alternatively, the discontinuous phase is aqueous and thecontinuous phase is an oil-based or lipidic medium.

Also alternatively, the discontinuous phase is aqueous and each dropletis surrounded by a single or multiple lipid bilayer (i.e. therebyforming a liposome), and the continuous phase is also aqueous.

Either or both of the discontinuous and continuous phases may comprisean emulsifier to stabilise the emulsion prior to the congregation of thenanoparticles. Suitable emulsifiers include lecithin, oleylamine, sodiumdeoxycholate, 1,2-distearyl-sn-glycero-3-phosphatidyl ethanolamine-N,stearylamine and 1,2-dioleoyl-3-trimethylammonium-propane. Preferably,the emulsifier is oleylamine which confers a positive charge to thedroplets.

The emulsifier will typically be provided in an amount in the range of0.0001 to 10 wt % of the emulsion, more preferably, in the range of 0.01to 1 wt % of the emulsion.

The active substance may be selected from nutriceutical substances,cosmetic substances (including sunscreens), pesticide compounds,agrochemicals and foodstuffs. However, preferably, the active substanceis selected from drug compounds. The active substance may be abiological agent such as a peptide, protein or nucleic acid (e.g.deoxyribonucleic acid (DNA)). Such biological agents are particularlysuitable for formulation within capsules comprising liposomes.

The nanoparticles may have hydrophilic or hydrophobic surfaces. However,when the discontinuous phase is an oil-based or lipidic medium,preferably the nanoparticles will have hydrophilic surfaces. In onepreferred embodiment, the droplets will be coated with a single layer,or multiple layers, of hydrophilic nanoparticles. However, in anotherpreferred embodiment, the droplets will be coated with at least twolayers of nanoparticles, the inner layer of nanoparticles havinghydrophobic surfaces while the outer layer of nanoparticles havehydrophilic surfaces.

The nanoparticles may be positively or negatively charged.

Preferably, said nanoparticles have an average diameter of 5-2000 nm,more preferably 20-80 nm, and most preferably about 50 nm. Also,preferably, the size of the nanoparticles will be such that the ratio ofnanoparticle size to the size of the nanoparticle-coated droplets (i.e.capsules) is in the range of 1:4 to 1:20 and, more preferably, is about1:10.

Preferably, the nanoparticles are composed of silica, howevernanoparticles composed of other substances (e.g. titania and latex) arealso suitable.

Congregation of the nanoparticles (e.g. by self-assembly and/oradsorption) at the phase interface results in the coating of the surfaceof the droplets in at least one layer of nanoparticles such thatsufficient structural integrity is provided to the droplets so that theymay withstand removal of the continuous phase to produce a driedformulation. By “structural integrity”, it is to be understood that thecapsules substantially retain the active substance (i.e. the capsules donot exhibit substantial leaching of the active substance) and do notsubstantially coalesce with one another to form larger capsules overtime. To achieve such structural integrity may require providing thenanoparticles to the two-phase liquid system within a particularconcentration range.

Preferably, the congregation of the nanoparticles at the phase interfaceoccurs in the presence of an amount of electrolyte suitable to enhancethe congregation of the nanoparticles at the phase interface. The amountof the electrolyte will typically be at least 0.5×10⁻⁴ M, preferably, atleast 1×10⁻³ M. However, preferably, the concentration of electrolytewill be no more than 1×10⁻¹ M.

Preferably, the electrolyte is NaCl.

The removal of the continuous phase is a drying step which may beperformed using a rotary evaporator. Alternatively, the removal of thecontinuous phase may be performed by freeze drying, spray drying orfluidised bed procedures.

Following step (ii) but prior to the drying step, additionalnanoparticles may be added to the two-phase liquid system, if desired.

The capsules of the dried formulation may be readily re-dispersed into aliquid to re-form a two-phase liquid system. In particular, there-dispersed capsules may form a capsule-based emulsion (which might bea capsule-based liposome emulsion) which is substantially identical orsimilar in composition to that from which the dried formulation wasprepared after storage at room temperature for 24 hours, and morepreferably, after storage at room temperature for 2 months.“Substantially identical or similar” in this context is intended to meanthat the average diameter size of the capsules is the same or variesfrom the original capsules by no more than a factor of about 4 times(i.e. the average diameter size of the re-dispersed capsules is no morethan 4 times greater in size or 4 times less in size than the originalcapsules). Further, preferably, few (if any) of the re-dispersedcapsules have a diameter size greater than 10 μm; for example,preferably less than 5% of the re-dispersed capsules, by volume, have adiameter size of greater than 10 μm). The re-dispersed capsules arestable and typically show no substantial degradation after 24 hoursstorage at room temperature (i.e. after 24 hours, the average diametersize of the re-dispersed capsules remains at no more than 4 timesgreater in size or 4 times less in size than the original capsules, andpreferably less than 5% of the re-dispersed capsules, by volume, have adiameter size of greater than 10 μm).

In a variation of the present invention, prior to the removal of thecontinuous phase, the capsules may be provided with a polymer layeraround the periphery to modify the interfacial properties of thecapsule.

In a further variation, the discontinuous phase may, optionally, becross-linked or otherwise comprise a gelling material so as to form amatrix. While re-dispersed capsules from dried formulations produced inaccordance with the present invention are permeable (i.e. thenanoparticle coating will be porous), and thereby typically showcontrolled release of the active substance at rates dependent upon thedegree of permeability (e.g. a capsule with a lower degree ofpermeability (i.e. a “semi-permeable” capsule), will show sustainedrelease of the active substance), the inclusion of a cross-linked orgelled matrix within the discontinuous phase can be used to providefurther control to the release of the active substance from thecapsules, particularly sustained release.

In a still further variation of the present invention, the nanoparticlesprovided to the two-phase liquid system congregate at the phaseinterface while the continuous phase is being removed (i.e. during thedrying step).

Thus, in a second aspect, the present invention provides a method ofproducing a dried formulation for an active substance, said methodcomprising the steps of:

-   (i) dispersing a discontinuous phase comprising an active substance    into a continuous phase so as to form a two-phase liquid system    comprising droplets of said discontinuous phase, each of said    droplets having, at its surface, a phase interface; and-   (ii) removing the continuous phase to produce a dried formulation,    during which nanoparticles provided to said two-phase liquid system    congregate at the phase interface to coat said surface of the    droplets in at least one layer of said nanoparticles, wherein said    at least one layer of nanoparticles provides sufficient structural    integrity to the droplets to withstand the removal of the continuous    phase.

The method of the second aspect is particularly suitable wherein thedroplets are negatively charged, and the nanoparticles to be used arenegatively charged, hydrophilic nanoparticles.

In a third aspect, the present invention provides a dried formulationfor an active substance, said formulation comprising droplets formed bydispersing a discontinuous phase comprising an active substance into acontinuous phase so as to form a two-phase liquid system, wherein eachdroplet is coated in at least one layer of said nanoparticles and thecontinuous phase has been removed.

The formulation comprises droplets formed by dispersing a discontinuousphase into a continuous phase to form a two-phase liquid system. As withthe methods of the first and second aspects of the present invention,preferably the discontinuous phase is an oil-based or lipidic medium andthe continuous phase is aqueous. Either or both of the discontinuous andcontinuous phases may comprise an emulsifier (e.g. lecithin) tostabilise the emulsion prior to coating with at least one layer ofnanoparticles.

The active substance may be selected from those mentioned above.

Preferably, said nanoparticles have an average diameter of 5-2000 nm,more preferably 20-80 nm, and most preferably about 50 nm. Also,preferably, the size of the nanoparticles will be such that the ratio ofnanoparticle size to capsule size is in the range of 1:4 to 1:20 and,more preferably, is about 1:10.

Preferably, the nanoparticles are composed of silica, howevernanoparticles composed of other substances (e.g. titania and latex) arealso suitable.

The capsules of the dried formulation may be readily re-dispersed into aliquid to re-form a two-phase liquid system. In particular, there-dispersed capsules may form a capsule-based emulsion which issubstantially identical or similar in composition to that from which thedried formulation was prepared.

In variations of the formulation of the present invention, the capsulesmay be provided with a polymer layer around the periphery to modify theinterfacial properties of the capsule. Also, the discontinuous phasemay, optionally, be cross-linked or otherwise comprise a gellingmaterial so as to form a matrix, which may enable controlled release ofan active substance (i.e. sustained release) from the capsules.

The present invention provides a method for producing dried formulationsof nanoparticle-coated capsules comprising a drug compound. An advantageof such formulations is that the dried capsules (e.g. in the form of adry powder), have a long shelf life and do not exhibit substantialleaching of the drug compound over times that drug formulations arecommonly stored (e.g. 1 to 9 months). In addition, the capsules have alow propensity to coalescence. The dried capsules can be readilyre-dispersed into a liquid to re-form a stable emulsion, therebyproviding a useful drug formulation for the pharmaceutical industry. Thecapsules can be readily stored and/or transported dry.

In addition, the nanoparticle coating on the droplets of the capsulescan protect labile active substances (i.e. chemically unstablesubstances) from degradation caused by acidity (i.e. low pH), oxidationand crystallisation, etc. The nanoparticle coating is also resistant towater (i.e. the nanoparticle coat does not substantially expand ordegrade in the presence of water).

The ability to protect the active substances from the degradativeeffects of acidity, makes the dried formulation of the present inventionparticularly useful in the oral administration of labile drug compounds(i.e. where it is desirable that the drug compound be protected from thehigh acidity of the stomach before reaching the small intestine wherethe drug compound may be adsorbed into the bloodstream).

In a further aspect, the present invention provides a formulation for anactive substance, said formulation comprising droplets formed bydispersing a discontinuous phase comprising an active substance into acontinuous phase so as to form a two-phase liquid system, wherein eachdroplet is coated in at least one layer of said nanoparticles.

The formulation of the further aspect can be dried or, otherwise, can beused in its liquid form.

In one preferred embodiment of the formulation of the further aspect,the capsules are provided with a polymer layer around the periphery tomodify the interfacial properties of the capsule. In this way, thecapsules may be made to be “lipoadhesive”, particularly if the polymerlayer has adhesive properties with lipid-like surfaces. One significantaspect of this is that the capsules can be then be engineered to adhereto particular sites in vivo (e.g. mucoadhesive polymer layers facilitateadhesion to mucous membranes), thereby ensuring long contact times andeffective transport of the active substance. This can be particularlyuseful for the delivery of poorly soluble drug compounds to variousparts of the gastrointestinal tract (i.e. to improve bioavailability).It may also be used to facilitate delivery of an active substance to themouth.

The combination of polymers and nanoparticles at the capsule surface canlead to further controlled release properties.

The invention will be generally discussed hereinafter in relation todrug delivery from emulsions but it is not so restricted and asmentioned above, nanoparticles may congregate at the phase interface ofother suitable vehicles (e.g. liposomes and solid particles).

Throughout this specification and the claims that follow unless thecontext requires otherwise, the words “comprise” and “include” andvariations such as “comprising” and “including” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that suchprior art forms part of the common general knowledge.

Specific embodiments of the invention will now be described in somefurther detail with reference to and as illustrated in the accompanyingfigures. These embodiments are illustrative, and not meant to berestrictive of the scope of the invention. Suggestions and descriptionsof other embodiments may be included within the scope of the inventionbut they may not be illustrated in the accompanying figures oralternatively features of the invention may be shown in the figures butnot described in the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

An illustrative embodiment of the present invention is discussedhereinafter with reference to the accompanying drawings wherein:

FIG. 1 is a cross-sectional schematic of an emulsion known in the art;

FIG. 2 is a cross-sectional schematic of a nanoparticle-stabilisedemulsion according to the present invention;

FIG. 3 is a graph to show adsorption isotherms of hydrophilic silicananoparticles assembling at the oil water interface;

FIG. 4 is a graph to show adsorption isotherms of hydrophobic silicananoparticles assembling at the oil water interface;

FIG. 5 is a flow chart showing the steps involved in obtaining the drycapsules of the present invention;

FIG. 6 is a schematic of the processes involved in obtaining thecapsules of the present invention as well as showing the re-dispersionof the capsules;

FIG. 7a shows the emulsion droplet size range;

FIG. 7b is the tabular form of FIG. 7a , showing the emulsion dropletsize range;

FIG. 8 shows the average diameter of the silica nanoparticles innanometres; and

FIG. 9 shows the long-term physical stability of negatively chargedemulsions in the presence of increasing concentrations of hydrophilicsilica nanoparticles.

DESCRIPTION OF PREFERRED EMBODIMENT

FIG. 1 is a cross-sectional schematic of a two-phase liquid systemreferred to as an emulsion having a discontinuous oil phase in the formof droplets 10 dispersed in a continuous aqueous phase 12, therebydefining a phase interface 14. After a period of time, adjacent oildroplets 10 will coalesce (the beginning of phase separation) to formlarger oil droplets. If an emulsion is not stabilised by an emulsifierlocalised in the thin film 16, then coalescence of the emulsion willoccur within minutes. Eventually the oil phase 10 and aqueous phase 12will have completely separated into the two component phases (oil andwater).

FIG. 2 shows the system of FIG. 1 where droplets 10 have been stabilisedby nanoparticles 18 at the interface 14. Two otherwise immiscibleliquids (10 and 12) have thereby formed a stabilised emulsion (nb. FIG.2 is a schematic and nanoparticles 18 are not drawn to scale withrespect to droplets 10).

In the preferred embodiment, as described above, the discontinuous phaseis an oil-based or lipidic medium and the continuous phase is aqueous.However, the discontinuous phase may be an aqueous phase dispersed in anoil-based or lipidic medium. Further, the discontinuous phase may beaqueous and each droplet surrounded by a single or multiple lipidbilayer (i.e. thereby forming a liposome), and the continuous phase isalso aqueous.

In order to improve biocompatibility of the emulsion, the oil phase canbe a fatty-food simulant such as a triglyceride (e.g. Miglycol 812™).Alternatively, the oil phase can be a silicone such aspolydimethlysiloxane (PDMS), or any other oily medium which will form anemulsion with an aqueous phase.

Nanoparticles 18 are dispersed in a liquid by sonication and provided tothe emulsion in order to coat each droplet 10 in at least one layer ofnanoparticles. In a preferred embodiment, the liquid dispersioncomprises 1% by weight (1 wt %) of nanoparticles in an aqueous medium(i.e. 1 g of nanoparticles per 100 ml). However, other weight %dispersions can be usefully employed. Upon addition, the nanoparticlescongregate at the phase interface 14 (e.g. by self-assembly).Alternatively, rather then being added to the preformed emulsion,nanoparticles 18 can be first dispersed in either phase (i.e. the oil oraqueous phase) or both phases (i.e. the oil and the water phase) and, asan emulsion is formed, nanoparticles 18 will congregate at the phaseinterface 14. Nanoparticles 18 form at least a partial coating over thesurface of droplets 10 (phase interface 14). The resultingnanoparticle-coated droplet is referred to as a capsule 20.

Preferably, the ratio of nanoparticle size to capsule size is between1:4 and 1:20. The nanoparticles 18 which stabilise the emulsion may havean average diameter in the range 5 nm-2000 nm and may be made from anysuitable material (e.g. titania or latex). Preferably, the nanoparticlesare silica nanoparticles having an average diameter of between 20-80 nm.In the preferred embodiment, the nanoparticles have an average diameterof approximately 50 nm and the capsule diameter size ranges between200-850 nm with an average capsule size of approximately 500 nm. Theapproximate ratio of nanoparticle to capsule size is therefore,preferably about 1:10.

In a preferred embodiment, the nanoparticles are Aerosil® silicananoparticles (Degussa AG, Dusseldorf, Germany). The surfaces ofnanoparticles 18 may be chemically or physically modified tohydrophobise the nanoparticles 18.

Capsule 20 has a liquid core 22 (the discontinuous phase) which maycomprise or contain active substance 24. Preferably, the liquid core 22is a hydrophobic or lipidic medium and contains a lipophilic activesubstance 24 therein. It is an option, however, that the liquid core 22is aqueous and has a hydrophilic active substance 24 dissolved therein.In FIG. 2, the cross-sectional depiction shows active substance 24. Theactive substance may be any substance which is required to be protectedand/or delivered by capsule 20. The active substance may be selectedfrom nutriceutical substances, cosmetic substances (includingsunscreens), pesticide compounds, agrochemicals and foodstuffs. In thepreferred embodiment, the active substance 24 is a drug compound. Theactive substance 24 may be wholly or partially soluble or dispersiblewithin liquid core 22. Also, the oil phase may, optionally, becross-linked or otherwise comprise a gelling material so as to form amatrix which can enable controlled release of an active substance (i.e.sustained release) from the capsules.

It is an option that the outer surface of the capsules 20 be coated witha layer that improves the interfacial properties of the capsules. Forexample, in drug delivery, capsules 20 may be further coated with apolymer layer around the periphery of capsule 20 to increase thebioadhesivity of the capsule to cells within the body. Such a layer maycomprise a polymer selected from methylcellulose,hydroxypropylcellulose, ethylcellulose, polyethyleneglycols, chitosan,guar gum, alginates, eudragit and pemulen, etc. Other coatings aroundthe capsule 20 which improve or modify the interfacial properties of thecapsule may be used. An example of the preparation of a coated capsuleis given in Example 5.

Drying the Capsules

A delivery system which is dry and can be transported, stored and/oradministered as a powder is an advantage in many industries, such as thepharmaceutical industry, since dry powder formulations usually have ahigher active substance content compared with an aqueous formulation.This means that less volume of the delivery system is required foradministration of an effective amount of active substance. The increasein active substance content in dry formulations is mainly due to theelimination of unnecessary liquids.

FIG. 5 is a flow chart outlining the processes involved in obtaining thedried formulation. In step 26, the amount (i.e. volume of nanoparticle 1wt % dispersion) of nanoparticles and, optionally, the properties ofnanoparticles 18, provided to the emulsion, are selected or otherwisecontrolled so that capsules 20 can withstand the removal of thecontinuous phase during a subsequent drying step (discussed furtherbelow). The nanoparticles should provide sufficient structural integrityto the coated droplets (capsules) to enable the subsequent removal ofthe continuous phase to produce the dry formulation. A capsule having“structural integrity” substantially retains the active substance withinits core and does not exhibit substantial leaching of the activesubstance and also does not substantially coalesce with other capsulesto form larger capsules over time. To achieve such structural integritymay require providing the nanoparticles to the two-phase liquid systemwithin a particular concentration range as described below.

The emulsion can be dried by any suitable method, for example freezedrying, spray drying or fluidised bed techniques. In step 28, theemulsion is dried by spray-drying and the resulting dried capsules arecollected in a suitable vessel.

FIG. 6 depicts the dried capsules 20 in vessel 33. Dried capsules 20have nanoparticles 18 congregated at their surface 14. Once dried, it isan option that dried capsules 20 are delivered in dry form (step 32).Dry formulations have increased active substance loading, therebyreducing the amount of formulation that is required. A further advantageis that the risk of microbial growth, which can cause serious infectionsor spoilage, is reduced in dry formulations compared with liquidformulation.

Not all capsules formed in the wet phase are able to be dried (i.e. somecapsules lack the abovementioned structural integrity). Table 1 belowshows the results of a number of experiments in which the capsulescollapsed during the drying step.

Table 1 shows that of the 27 different tested variations (usinghydrophilic silica nanoparticles with an average diameter of about 50nm, and an oil-based discontinuous phase stabilised with lecithin(negatively charged oil droplets) or oleylamine (positively chargeddroplets) within an aqueous continuous phase), 19 combinations formedcapsules which maintained their structural integrity during removal ofthe continuous phase. In the first six rows of the table, a dry powderof capsules could not be obtained due to loss of structural integrityand subsequent degradation of capsules. The experiments shown in rowsG-ι, show the oil to nanoparticle mass ratios which formed dry capsules.

It can be seen that an oil to nanoparticle mass ratio of at least 1:0.05was required in order to be able to produce dried capsules withpositively charged droplets, and that an oil to nanoparticle mass ratioof at least 1:0.2 was required in order to be able to produce driedcapsules with negatively charged droplets.

TABLE 1 Emulsion and hydrophilic silica nanoparticle amounts to producedry capsules. The oil droplets were stabilised with lecithin oroleylamine. Volume [NaCl] of Volume in overall emulsion of Mass mixtureOverall Ratio of (10 wt % particles Mass of volume mixtureOil(wt):particles Row Label oil) (1 wt %) of oil particles (1 ×10^(−x)M) volume (wt) A Dried 10 ml  10 ml    1 g  0.1 g 10⁻⁴  20 ml1:0.1 capsules not obtained B Dried 10 ml  10 ml    1 g  0.1 g 10⁻²  20ml 1:0.1 capsules not obtained C Dried 10 ml  5 ml   1 g 0.05 g 10⁻⁴  20ml 1:0.05 capsules not obtained D Dried 10 ml  5 ml   1 g 0.05 g 10⁻² 20 ml 1:0.05 capsules not obtained E Dried 10 ml  1 ml   1 g 0.01 g10⁻⁴  20 ml 1:0.01 capsules not obtained F Dried 10 ml  1 ml   1 g 0.01g 10⁻²  20 ml 1:0.01 capsules not obtained G Dried 1 ml 10 ml  0.1 g 0.1 g 10⁻⁴  20 ml 1:1 capsules obtained H Dried 1 ml 10 ml  0.1 g  0.1g 10⁻²  20 ml 1:1 capsules obtained I Dried 1 ml 10 ml  0.1 g  0.1 g10⁻¹  20 ml 1:1 capsules obtained J Dried 1 ml 10 ml  0.1 g  0.1 g 10⁻⁴ 11 ml 1:1 capsules obtained K Dried 1 ml 5 ml 0.1 g 0.05 g 10⁻⁴  20 ml1:0.5 capsules obtained L Dried 1 ml 5 ml 0.1 g 0.05 g 10⁻²  20 ml 1:0.5capsules obtained M Dried 1 ml 5 ml 0.1 g 0.05 g 10⁻¹  20 ml 1:0.5capsules obtained N Dried 1 ml 5 ml 0.1 g 0.05 g 10⁻⁴  6 ml 1:0.5capsules obtained O Dried 1 ml 5 ml 0.1 g 0.05 g 10⁻²  6 ml 1:0.5capsules obtained P Dried 1 ml 5 ml 0.1 g 0.05 g 10⁻⁴  10 ml 1:0.5capsules obtained Q Dried 1 ml 5 ml 0.1 g 0.05 g 10⁻²  10 ml 1:0.5capsules obtained R Dried 1 ml 5 ml 0.1 g 0.05 g 10⁻¹  10 ml 1:0.5capsules obtained S Dried 5 ml 5 ml 0.5 g 0.05 g 10⁻⁴ 100 ml 1:0.1capsules obtained T Dried 5 ml 15 ml  0.5 g 0.15 g 10⁻⁴ 100 ml 1:0.3capsules obtained U Dried 5 ml 25 ml  0.5 g 0.25 g 10⁻⁴ 100 ml 1:0.5capsules obtained V Dried 5 ml 50 ml  0.5 g  0.5 g 10⁻⁴ 100 ml 1:1capsules obtained W Dried 5 ml 95 ml  0.5 g 0.95 g 10⁻⁴ 100 ml 1:2capsules obtained X Dried 25 ml  25 ml  2.5 g 0.25 g 10⁻⁴ 100 ml 1:0.1Capsules obtained Y Dried 25 ml  50 ml  2.5 g  0.5 g 10⁻⁴ 100 ml 1:0.2capsules obtained Z Dried 25 ml  95 ml  2.5 g 0.95 g 10⁻⁴ 100 ml 1:0.4capsules obtained α Dried 50 ml  50 ml    5 g  0.5 g 10⁻⁴ 100 ml 1:0.1capsules obtained β Dried 15 ml  7.5 ml   1.5 g 0.075 g  10⁻⁴ 100 ml1:0.05 capsules obtained Ψ Dried 15 ml  15 ml  1.5 g 0.15 g 10⁻⁴ 100 ml1:0.1 capsules obtained δ Dried 15 ml  30 ml  1.5 g  0.3 g 10⁻⁴ 100 ml1:0.2 capsules obtained ε Dried 25 ml  12.5 ml   2.5 g 0.125 g  10⁻⁴ 100ml 1:0.05 capsules obtained φ Dried 25 ml  25 ml  2.5 g 0.25 g 10⁻⁴ 100ml 1:0.1 capsules obtained γ Dried 25 ml  47.5 ml   2.5 g 0.475 g  10⁻⁴100 ml 1:0.2 capsules obtained η Dried 50 ml  25 ml    5 g 0.25 g 10⁻⁴100 ml 1:0.05 capsules obtained ι Dried 50 ml  50 ml    5 g  0.5 g 10⁻⁴100 ml 1:0.1 capsules obtained

The capsules of experiments A to R, T to W, Y, Z, δ and γ were preparedfrom emulsions stabilised with lecithin (i.e. negatively chargeddroplets), while the capsules of experiments S, α to ψ, Σ, φ, η and ιwere prepared from emulsions stabilised with oleylamine (i.e. positivelycharged droplets). The capsules of experiments A to R were dried usingrotary evaporation, while the capsules of experiments S to ι were driedusing spray-drying.

Properties of Driable Capsules (1) Wettability of Nanoparticles

Nanoparticles 18 (e.g. silica nanoparticles) can be modified to behydrophobic. In a preferred embodiment, the surfaces of nanoparticles 18are modified with organosilanes (e.g. dimethylchlorosilane). Thecoalescence behaviour of capsule 20 is dependent upon the hydrophobicityor hydrophilicity of nanoparticles 18, as well as the coverage ofnanoparticles 18 at the emulsion droplet interface 14. At full orpartial coverage of hydrophilic nanoparticles 18, capsules 20 stilldisplay some degree of enlargement behaviour (i.e. the diameter of thecapsules increase during coalescence). In contrast, emulsion dropletscoated by more than one layer of hydrophobic nanoparticles 18 (underconditions of coagulation in the presence of high salt concentrations(e.g. 1×10⁻¹ M)), form stable flocculated networks rather than enlargedcapsules. Experiments have revealed that in the wet phase, it ispreferable that nanoparticles 18 have a hydrophobic surface whichreduces the occurrence of capsule 20 coalescence.

However, while hydrophobic nanoparticles form a stable wet phase capsulewith good protection of the active substance, further experiments haveindicated that hydrophilic nanoparticles better stabilise capsulesduring a drying phase. That is, the results of these experiments haveindicated that if the nanoparticles have a hydrophobic surface, then thecapsules may be unstable during the drying step. This may be due tomigration of the hydrophobic nanoparticles into the oil of the emulsiondroplet, resulting in instability of the capsules. It is an optiontherefore, that droplets are first coated with a hydrophobic layer ofnanoparticles to create a stable wet phase. The resulting capsules canthen be further coated by a hydrophilic layer of nanoparticles tostabilise the capsule during a drying phase. The further coat ofhydrophilic nanoparticles can be applied by adding the nanoparticles tothe continuous phase and allowing them to congregate onto the surface ofthe capsule while the wet phase is “standing” and/or during the dryingphase.

(2) Effect of Salt Concentration on Nanoparticle Congregation

Typical isotherms for hydrophilic silica nanoparticles adsorbing at amodel oil water interface 14 are shown in FIG. 3. It is clear that salt(electrolyte) addition dramatically increases nanoparticle adsorption.Preferably, the nanoparticles congregate at the phase interface in thepresence of an amount of electrolyte suitable to enhance thecongregation of the nanoparticles at the phase interface. The amount ofthe electrolyte will typically be less than 1×10⁻¹ M (preferably, atleast 1×10⁻³ M and more preferably, at least 0.5×10⁻⁴M). In thepreferred embodiment, NaCl is used, however it will be understood bypersons skilled in the art that any electrolyte may be used.

While not wishing to be bound by theory, it is considered that the freeenergy of nanoparticle adsorption increases significantly with saltaddition due to a reduction in the range of nanoparticle-droplet andnanoparticle-nanoparticle lateral electrostatic repulsion. In high saltconcentrations (e.g. 1×10⁻² and 1×10⁻¹ M NaCl), adsorption amounts forhydrophilic nanoparticles 18 correspond to approximately 75% and justover 100% of an equivalent hexagonally close-packed monolayer of hardspheres respectively. The fractional surface coverage is anapproximation calculated from the ratio of adsorbed amount ofnanoparticles 18 and the theoretical value for a hexagonally closepacked monolayer (i.e. 200 mg·m⁻² for 50 nm diameter nanoparticles).

(3) Effect of Charged Oil Droplets on Nanoparticle Congregation

It is an option that, prior to the addition of nanoparticles 18, anegatively charged phospholipid monolayer, such as lecithin or apositively charged oleylamine is used as a stabiliser to stabilise theoil droplets of the emulsion (emulsifier 14 is shown in FIG. 1). Bothlecithin and oleylamine are fat emulsifiers which help to preventdroplets 10 from coalescing before nanoparticles 18 congregate. Otherstabilisers similar to oleylamine, which are particularly useful in thepresent invention, include 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine-N, stearylamine and1,2-dioleoyl-3-trimethylammonium-propane.

Experiments have shown that negatively charged phospholipid stabilisedtriglyceride droplets do not strongly interact with hydrophilic silicananoparticles. This is evidenced by adsorption studies, freeze fractureSEM and is supported by EDAX surface elemental analysis. Positivelycharged oleylamine stabilised triglyceride droplets on the other hand,strongly interact with hydrophilic silica nanoparticles as evidence byadsorption studies, charge reversal and freeze-fracture SEM.

(4) Phase from which Nanoparticles Congregate

As stated above, the nanoparticles can be first dispersed in eitherphase (oil or water) and, as an emulsion is formed, the nanoparticleswill congregate at the phase interface.

Initial studies have shown that very few negatively chargednanoparticles from the aqueous phase (less than 5%) congregate at thedroplet surface of negatively charged droplets (e.g. droplets stabilisedwith lecithin), although greater levels of nanoparticle congregation hasbeen observed with droplets of silicone (i.e. PDMS). Positively chargeddroplets however, are coated by nanoparticles dispersed within theaqueous phase.

(5) Oil:Nanoparticle Mass Ratio

The oil (g) to nanoparticle (g) ratio plays an important role inpreparing capsules which can withstand the drying step (i.e. driablecapsules). That is, an oil to nanoparticle mass ratio of at least 1:0.02is considered to be necessary in order to produce dried capsules.However, preferably, an oil to nanoparticle mass ratio of at least1:0.05 and, more preferably, at least 1:0.2, is used.

Properties of Redispersible Capsules

The capsules are prepared so as to remain stable and do notsubstantially coalesce to form capsules with an increased diameter. Thepresent invention therefore has the advantage of maintaining the releaseprofile of the active substance contained within the capsule as well asmaintaining the small size of the capsules. The small size of thecapsules both increases surface area and allows the capsules to bedelivered to target areas which require a small capsule size (e.g. bloodcapillaries). Capsules 20 may therefore have a longer shelf life thanprior capsule formulations and can be stored and/or transported forlater use.

Preferably, the dried capsules 20 can be re-dispersed (shown by step 30)in a liquid (preferably water) to re-form a stabilised emulsifiedproduct. Not all dried capsules are satisfactorily re-dispersible andagain, the properties selected during capsule formation are important.Dried capsules in accordance with the present invention, however, can bemade to re-disperse in a liquid to form an emulsion which issubstantially identical or similar in composition to that from which thedried formulation was prepared. This means that the average capsulediameter size is the same or varies from the original capsule by no morethan a factor of about 4 times and, preferably, shows few (i.e. lessthan 5% by volume), if any, capsules with a diameter size of greaterthan 10 μm.

The re-constitutive properties following the re-dispersion of capsulesof Table 1 in phosphate buffer are shown in Table 2 below. Thereconstitution mark rates how similar the reconstituted emulsioncompared with the emulsion from which the capsules were dried.

The re-constitutive properties following re-dispersion of capsules ofTable 1 in acidic medium after 2 months of storage at room temperatureare shown in Table 3 below.

TABLE 2 Average capsule size and reconstitution rating followingre-dispersion of capsules (in phosphate buffer (pH = 7.2)) listed inTable 1. 0.01 g of powder was dissolved in 4 g of phosphate buffer 10⁻⁴Mafter 24 hours from drying (size measured using Malvern Mastersizer)Average Average Row drop size re-dispersed Vol % (from Oil:particlebefore drop size D (v, 0.9) above Reconstitution Table 1) ratio drying(μm) (μm) (μm) 10 μm mark G 1:0.1 1.04 1.27 — 2 Very good H 1:0.1 1.821.99 — 5 Good I 1:0.1 8.93 22.04 — 19 Poor J 1:0.1 13.67 28.89 — 24 PoorK 1:0.05 0.76 1.04 — 0.5 Excellent L 1:0.05 0.94 1.55 — 0.5 Excellent M1:0.05 12.99 56.6 — 87 Very poor N 1:0.05 26.75 47.3 — 89 Very poor O1:0.05 5.87 30.05 — 31 Very poor P 1:0.05 1.52 2.51 — 0 Excellent Q1:0.05 0.88 2.15 — 0 Excellent R 1:0.05 1.36 5.23 — 2 Very good S 1:0.1— — — — Oily paste T 1:0.3 — 25 88 98 Very poor U 1:0.5 — 0.52 0.65 0Excellent V 1:1 — 0.78 1.27 0 Excellent W 1:2 — 0.55 0.68 0 Excellent X1:0.1 — — — — Oily paste Y 1:0.2 — 40.3 131.12 98 Very poor Z 1:0.4 —1.02 16.96 95 Very poor α 1:0.1 — — — — Oily paste β 1:0.5 — 0.82 2.57 0Excellent Ψ 1:1 — 0.53 0.72 0 Excellent δ 1:2 — 0.72 1.17 0 Excellent ε1:0.5 — 0.46 0.66 0 Excellent φ 1:1 — 0.66 0.98 0 Excellent γ 1:2 — 0.580.78 0 Excellent η 1:0.5 — 0.46 0.66 0 Excellent ι 1:1 — 0.52 0.72 0Excellent control — — 0.67 0.92 0 Excellent (only silica)

TABLE 3 Average capsule size and reconstitution rating followingre-dispersion of the capsules in acidic media (pH = 2, adjusted withhydrochloric acid) after 2 months of storage (measured using MalvernMastersizer) Average D Vol % Row (from Oil:particle drop size (v, 0.9)above Reconstitution Table 1) ratio (μm) (μm) 10 μm mark S 1:0.1 — — —Oily paste T 1:0.3 54.6 133.6 98 Very poor U 1:0.5 4.7 10.1 Below 5 GoodV 1:1   2.39 7.42 Below 2 Very good W 1:2   0.55 0.68 0 Excellent X1:0.1 — — — Oily paste Y 1:0.2 105.2 167 98 Very poor Z 1:0.4 3 39.3 50Very poor α 1:0.1 — — — Oily paste control — 0.64 0.93 0 Excellent (onlysilica)

It is clear that the capsules of experiments U, V and W showed the bestre-dispersibility and reconstitution after 2 months of storage (nb.after 8 months of storage, the respective average drop size of U, V andW, were 4.34 μm, 2.59 μm and 1.65 μm, against 3.34 μm of the control).These capsules were produced in the presence of a relatively low amountof electrolyte (i.e. 1×10⁻⁴ M) and with an oil to nanoparticle massratio of at least 1:0.5. They were prepared from negatively charged oildroplets (stabilised with lecithin). It is considered that for suchnegatively charged oil droplets, an oil to nanoparticle ratio of atleast 1:0.2 is required to achieve droplets that are wholly coated innanoparticles.

On the other hand, for positively charged oil droplets (e.g. stabilisedwith oleylamine), it is considered that the droplets interact morestrongly with the nanoparticles and, therefore, the minimum oil tonanoparticle ratio is less; in particular, an oil to nanoparticle massratio of at least 1:0.05 is believed to be required to produce driedcapsules that can be re-dispersed to form a capsule-based emulsion whichis substantially identical or similar to that from which the driedformulation was prepared. This ratio is believed to result in theproduction of wholly coated droplets, however, it is preferable to usean oil to nanoparticle mass ratio of least 1:0.1.

The optimum ratio of nanoparticles (g/cm³) to lecithin (g/cm³) has beenfound to be 5:1 when nanoparticles congregate from the oil phase. Theoptimum ratio of nanoparticles (g/cm³) to oleylamine (g/cm³) has beenfound to be 1:10 when the nanoparticles congregate from either the oilor the water phase.

Example 1 a) Preparation and Characterisation of Emulsion Stabilised byLecithin

Lecithin (0.6 g) stabiliser was dissolved in triglyceride (Miglyol 812™)(10 g), and then added to water (total sample weight: 100 g) undermixing using a rotor-stator homogeniser (11,000 rpm, 10 minutes,pH=6.95±0.2). Alternatively, a high pressure homogeniser (5 cycles, 5mBars) can be used for production of the emulsion. After 24 hours, theemulsion was characterised in terms of size (laser diffraction MalvernMastersizer) and zeta potential (PALS). Droplet size distribution isshown in FIG. 9a and FIG. 9b . The droplet size ranges from 0.20-0.86μm.

b) Preparation of Nanoparticles

An aqueous dispersion of silica (Aerosil®) nanoparticles (1 wt %) wasprepared by sonication over at least a one hour period. FIG. 8 showsthat the average silica nanoparticle size was approximately 50 nm.

c) Capsule Formation

Emulsion formed in step (a) and nanoparticle dispersion (b) were mixedtogether. Subsequently, the volume of the mixture can be varied ifdesired by the addition of water. The salt concentration can be in therange of 1×10⁻⁴ to 1×10⁻¹.

d) Drying—Removal of Continuous Phase

In order to prepare dry emulsion powders, an emulsion and hydrophilicsilica dispersion were mixed in 20 ml vials and spray-dried underfollowing conditions: flow rate 5 ml/min., aspirator setting 10, airflow 0.6 m³/min, inlet temperature 160° C. and outlet temperature 85° C.

e) Redispersion and Characterisation of Capsules

Emulsions were redispersed in phosphate buffer (pH=7.2) and acidic media(pH=2) and the drop size distribution measured using a MalvernMastersizer and Malvern Zetasizer Nano. Dry emulsion powders were imagedusing SEM. Scanning electron microscopy was performed using a PhilipsSEM 515, operating at 15 kV. A thin layer of the samples was placed ondouble adhesive tape, sticked on SEM-stubs. The samples were coated withgold by a Balzers SCD 050, Balzer Union AG sputter prior to microscopy.The SEM images showed mono-disperse, smooth, spherical capsules whichmaintain their structural integrity even under the high vacuum requiredduring imaging. There was no evidence of capsule aggregation as is oftenobserved with SEM images of silica nanoparticles themselves. Thecapsules imaged had diameters within the range 100 to 300 nm indicatingthat the capsules are discrete oil droplets coated with at least onelayer of nanoparticles.

Example 2 a) Preparation of Emulsions

Simple Oil/Water lipid emulsions, containing 10% a 20% triglyceride(Miglyol® 812) as the oil phase, were prepared by high-pressurehomogenizer at 500-1000 bar and ambient temperature. Negatively andpositively charged emulsion oil droplets have been achieved by usinglecithin and oleylamine respectively, as emulsifiers initially added tothe oil phase. In the case of silica incorporated emulsions, silicananoparticles were added to the oil phase or aqueous phase of emulsions,initially stabilised by lecithin or oleylamine, and sonicated for 60minutes before homogenisation.

b) Size Analysis

Size measurements were carried out using laser diffraction by Malvern®Mastersizer (Malvern Instruments, UK) following appropriate dilution ofsamples with MiliQ water.

c) Freeze Fracture Scanning Electron Microscopy

A freeze-fracture SEM technique (Philips XL 30 FEG scanning electronmicroscope with Oxford Conn. 1500 cryotransfer system) was used to imagethe oil droplets. The precise method for effective imaging of thedroplets depends on the sample properties such as nanoparticle type andcoverage. Generally, the methodology contains emulsion cryofixation,fracturing, etching, platinum coating and imaging.

d) Physical Stability Tests

Long-term physical stability of emulsions was assessed by size analysisof emulsion droplets at determined for intervals up to 3 months storageat ambient temperature.

D (v, 0.5), D (v, 0.9) and specific surface area were considered asindicators of physical stability of emulsions.

e) Visual Inspection

Organoleptic characteristics (i.e. evidence of creaming and coalescence)of emulsions have been recorded in parallel with size analysis. (nb.since oil is less dense than the water each oil drop is prone tofloating upwards. This process is called creaming—the oil droplets willgradually form a dense layer at the top of the sample). The degree ofcreaming and phase separation is assessed by visual observation ofemulsions at given time intervals. Coalescence can be determined bymonitoring the mean droplet diameter of the emulsions during storageperiod. Organoleptically, the appearance of large oil droplets or alayer of free oil on the emulsion surface is the indicators of acoalesced emulsion.

Example 3 a) Long-Term Physical Stability

Long term physical stability of emulsions has been improved in thepresence of silica nanoparticles.

D (v, 0.9) of emulsions initially stabilised by lecithin, in the absenceand presence of silica nanoparticles has been shown in (FIG. 9). D (v,0.9) of silica-added emulsions was effectively unchanged during storageat room temperature for 3 months, whereas emulsions solely stabilised bylecithin have shown a 3-fold increase in D (v, 0.9).

Example 4

In this example, dried capsule formulations were prepared fromliposomes.

a) Liposome Preparation

0.3317 g lecithin and 0.1085 g cholesterol were dissolved in 20 mlchloroform and evaporated under vacuum. 20 ml MilliQ water was addedwith periodic sonication.

Liposomal dispersions were mixed with aqueous dispersions of silicananoparticles and spray-dried using standard procedure.

Sample 1: Liposome dispersion 5 g and 95 g of 1 wt % silica nanoparticledispersion;Sample 2: Liposome dispersion 5 g and 95 g 5 wt % silica nanoparticledispersion; andSample 3: Liposome dispersion 30 g and 70 g 5 wt % silica nanoparticledispersion.b) Reconstitution in MilliQ Water after 24 Hours

The reconstitution of liposome-based capsules is shown in Table 4 below.The dried liposome capsules showed good re-dispersion properties, withthe size distribution of the re-dispersed capsules being within therange of 0.5 to 5 μm.

TABLE 4 z-average drop Polydispersibility Zeta potentials Sample size(μm) index (PDI) (mV) 1 5.1 0.305 −4.94 ± 5.5  2 3.44 1.000 −19.4 ± 16.43 2.43 0.2 −26.1 ± 21.8

Example 5 a) General Preparation Method: Miglyol 10 g Lecithin 0.6 g orOleylamine 1 g Silica 0.2-0.5 g

Polymer aqueous dispersion (hydroxypropyl methyl cellulose 1 wt % orchitosan 0.5 wt % or carbomer 0.1 wt %) to 100.0

Lecithin or oleylamine is dissolved in Miglyol and silica is added andredispersed in Miglyol. After polymer dispersion addition, the mixtureis sonicated for 40 minutes and spray dried using standard procedures.

Samples were investigated for re-dispersibility in phosphate buffer,pH=7.2 using Malvern Zetasizer Nano after 24 hours storage at RT.

The re-dispersibility of samples is shown in Tables 5 to 10 (where PDIis the polydispersibility index):

i) Formulation 1: Migliol 10 g Oleylamine 1 g Silica 0.2 g

Polymer aqueous dispersion (hydroxypropylmethyl cellulose 1 wt %) to100.0

TABLE 5 Before Spray Drying dry powder re-dispersion in buffer z-averageZeta Zeta drop size potentials z-average potentials (μm) PDI (mV) dropsize PDI (mV) 0.364 0.375 +35.4 ± 5.24 0.932 0.123 +19.9 ± 7.03

ii) Formulation 2: Migliol 10 g Oleylamine 1 g Silica 0.5 g

Polymer aqueous dispersion (hydroxypropylmethyl cellulose 1 wt %) to100.0.

TABLE 6 Before Spray Drying dry powder re-dispersion in buffer z-averageZeta Zeta drop size potentials z-average potentials (μm) PDI (mV) dropsize PDI (mV) 0.324 0.445 +35.5 ± 8.54 1.05 0.123 +18.8 ± 10.2iii) Formulation 3:

Migliol 10 g Lecithin 0.6 g Silica 0.5 g

Polymer aqueous dispersion (hydroxypropylmethyl cellulose 1 wt %) to100.0.

TABLE 7 Before Spray Drying dry powder re-dispersion in buffer z-averageZeta Zeta drop size potentials z-average potentials (μm) PDI (mV) dropsize PDI (mV) 0.451 0.449 −6.02 ± 18 2.16 0.385 −10.1 ± 9.31

iv) Formulation 4: Migliol 10 g Oleylamine 1 g Silica 0.5 g

Polymer aqueous dispersion (carbomer 0.1 wt %) to 100.0.

TABLE 8 Before Spray Drying dry powder re-dispersion in buffer z-averageZeta Zeta drop size potentials z-average potentials (μm) PDI (mV) dropsize PDI (mV) 0.618 0.519 −58.5 ± 10.1 1.9 0.907 −29 ± 14.3

v) Formulation 5: Migliol 10 g Lecithin 0.6 g Silica 0.5 g

Polymer aqueous dispersion (carbomer 0.1 wt %) to 100.0.

TABLE 9 Before Spray Drying dry powder re-dispersion in buffer z-averageZeta Zeta drop size potentials z-average potentials (μm) PDI (mV) dropsize PDI (mV) 0.545 0.432 −51.2 ± 5.13 2.8 1.000 −25.8 ± 15.6

vi) Formulation 6: Migliol 10 g Oleylamine 1 g Silica 0.5 g

Polymer aqueous dispersion (chitosan 0.5 wt %) to 100.0.

TABLE 10 Before Spray Drying dry powder re-dispersion in bufferz-average Zeta Zeta drop size potentials z-average potentials (μm) PDI(mV) drop size PDI (mV) 0.556 0.497 +73.3 ± 12.5 1.53 0.450 +48.5 ± 4.8

Example 6

In this example, formulations of dried capsules were produced usingoleylamine as an emulsion stabiliser and tested for re-dispersion andreconstitution after 24 hours and 3 months storage at room temperature.

a) Preparation and Characterisation of Emulsion Stabilised by Oleylamine

Oleylamine (1.0 g) stabiliser was dissolved in triglyceride (Miglyol812™) (10 g), and then added to water (total sample weight: 100 g).Emulsion was produced using high pressure homogenizer (5 cycles, 5 mBarspressure). After 24 hours, the emulsion was characterised in terms ofsize (laser diffraction Malvern Mastersizer) and zeta potential (PALS).The droplet size ranges from 0.20-1.5 μm.

b) Preparation of Nanoparticles

An aqueous dispersion of silica (Aerosil®) nanoparticles (1 wt %) wasprepared by sonication over at least a one hour period. FIG. 8 showsthat the average silica nanoparticle size was approximately 50 nm.

c) Capsule Formation

Emulsion formed in step (a) and nanoparticle dispersion (b) were mixedtogether in the ratios shown in Table 11 below. Subsequently, the volumeof the mixture can be varied if desired by the addition of water. Thesalt concentration can be in the range of 1×10⁻⁴ to 1×10⁻¹.

d) Drying—Removal of Continuous Phase

In order to prepare dry emulsion powders, an emulsion and hydrophilicsilica dispersion were mixed in 20 ml vials and spray-dried underfollowing conditions: flow rate 5 ml/min., aspirator setting 10, airflow 0.6 m³/min, inlet temperature 160° C. and outlet temperature 85° C.

e) Redispersion and Characterisation of Capsules

Emulsions were redispersed in phosphate buffer (pH=7.2) and acidic media(pH=2) and the drop size distribution was measured using a MalvernMastersizer and Malvern zetananosizer. Results are shown in Table 11.

TABLE 11 Ratio of oil Average drop Average drop (wt):particles sizeafter 24 size after 3 Sample (wt) hours (μm) months (μm) 1 1:0.1 3.652.5 2 1:0.3 11.5 6.17 3 1:0.5 12.66 5.25 4 1:1   6.84 3.31 5 1:2   6.376.7

Modifications and variations such as would be apparent to personsskilled in the art are deemed to be within the scope of the presentinvention. For example, although the invention is generally discussedwith reference to emulsion droplets, the techniques discussed cangenerally be applied to liposomes, other vesicle systems and othersimilar vehicles. For example, at least one layer of nanoparticles maycongregate at the phase interface of the lipid layer of a vesicle andthe continuous phase in which the vesicle is dispersed.

1. A dried formulation for an active substance produced by spray- orfreeze-drying a dispersion of liposomes comprising a single or multiplelipid bilayer, surrounding an aqueous phase comprising said activesubstance, wherein said liposomes are coated with at least one layer ofnanoparticles congregated at a phase interface of said liposomes.
 2. Theformulation of claim 1, wherein the active substance is selected fromthe group consisting of nutriceutical substances, cosmetic substancesand drug compounds.
 3. The formulation of claim 1, wherein the activesubstance is a biological agent.
 4. The formulation of claim 3, whereinthe biological agent is selected from the group consisting of a peptide,a protein and a nucleic acid.
 5. The formulation of claim 1, wherein thenanoparticles have hydrophilic surfaces.
 6. The formulation of claim 1,wherein the nanoparticles are silica nanoparticles.
 7. The formulationof claim 6, wherein the nanoparticles are silica nanoparticles.
 8. Theformulation of claim 1, wherein the nanoparticles have an averagediameter in the range of 5-80 nm.
 9. The formulation of claim 8, whereinthe nanoparticles have an average diameter of about 50 nm.
 10. Theformulation of claim 1, wherein the single or multiple lipid bilayercomprises lecithin.
 11. The formulation of claim 1, wherein the singleor multiple lipid bilayer comprises lecithin and cholesterol.